A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies. Supplementary Information The online version contains supplementary material available at 10.1007/s11274-024-04124-6.


Introduction
Integrative and Conjugative Elements (ICEs) are Mobile Genetic Elements capable of stable integration into bacterial genome and intercellular transposition to a new genome by conjugal transfer to a broad range of hosts (Mullany et al. 2002).These characteristics make ICEs suitable tools for genetic manipulation of a large variety of transformable and non-transformable bacteria (Pozzi et al. 1988;Manganelli Mariana Tirziu and Lorenzo Colombini contributed equally to this work. et al. 1998).ICEs can be used for introduction of heterologous DNA into recipient hosts, for reintroduction of in vitro mutagenized DNA in the original host, physiological studies, and complementation assays (Smith and Clewell 1984;Choi, Kyoung-Hee and Kim, Kang-Ju 2009;).ICE Tn5253 (formerly called Ω6001) is a composite element found in Streptococcus pneumoniae BM6001 (Dang- Van et al. 1978;Colombini et al. 2023a) that contains Tn5251, an ICE essentially identical to the Enterococcus faecalis Tn916 carrying the tetracycline resistance gene tet(M) (Provvedi et al. 1996;Santoro et al. 2010Santoro et al. , 2014;;Iannelli et al. 2014).Tn5253 transfers in different bacterial species with a frequency of conjugation up to 6.7 × 10 − 3 transconjugants per donor (Santoro et al. 2010(Santoro et al. , 2018)).Chromosomal integration of Tn5253 occurs downstream of a conserved 11-bp sequence of the essential rbgA gene in S. pneumoniae and in other bacterial species (Santoro et al. 2021).The insertion vector pDP36 was designed to integrate heterologous DNA into the chromosome of streptococci carrying Tn5253 and the resulting recombinant ICE was transferred to streptococci and enterococci by conjugation (Pozzi et al. 1988;Oggioni and Pozzi 1990).Vector pSMB47, a derivative of pDP36, carrying two non-contiguous DNA fragments of Tn5251 was constructed and used to introduce heterologous DNA in Bacillus subtilis and E. faecalis strains carrying Tn916 (Manganelli et al. 1998).A host-vector system for construction, chromosomal integration, and expression of translational-fusions with the Streptococcus pyogenes emm6.1 gene, encoding the surface protein M6, was developed and used for surface display of several heterologous proteins in Streptococcus gordonii (Pozzi et al. 1992a, b;Oggioni and Pozzi 1996;Oggioni et al. 1999).The system is based on an insertion vector which allows for chromosomal integration of fusion genes under the control of a strong constitutive promoter in a specifically engineered S. gordonii transformation recipient.S. pneumoniae is an important human pathogen, naturally competent for genetic transformation, easy to genetically manipulate, thus representing a model for the study of bacterial genetics and pathogenesis (Santoro et al. 2019).Several genetic tools have been developed to manipulate S. pneumoniae, among these controlled gene expression systems represent a powerful tool to study pneumococcal physiology.The S. pneumoniae Chromosomal Expression Platform (CEP), permits expression of a target gene under the control of a maltose-inducible promoter (Guiral et al. 2006a, b).This system, located in a silent transcriptional site and based on a preassembled insertion vector, was used for controlled expression of the luciferase gene and the competence comDE operon.The Pneumococcal Engineering Platform (PEP) is based on insertion vectors carrying an IPTG-inducible promoter, able to integrate in the majority of S. pneumoniae genomes.This system was efficiently utilized to express newly described fluorescent proteins (Keller et al. 2019).The nisin-controlled gene expression system (NICE), based on nisin biosynthesis autoregulation, was developed in L. lactis and used to express genes of different origins in different hosts including S. pneumoniae.The system relies on target gene cloning downstream of the nisin inducible PnisA promoter via the two-component system nisRK encoding the histidine kinase NisK and the response regulator NisR (Kuipers et al. 1993(Kuipers et al. , 1995;;De Vos et al. 1995;Zhou et al. 2006).In this study, we developed a chromosomally stable host-vector expression system, inducible by nisin in S. pneumoniae.The system is constituted by an insertion vector designed to integrate in the pneumococcal ICE Tn5253 and is therefore transferable by conjugation to a broad range of bacterial species, making the system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Bacterial strains, growth conditions, and minimal inhibitory concentration (MIC) determination
Bacterial strains used in this work and their relevant properties are reported in Table 1.Streptococci and enterococci strains were routinely grown at 37°C in tryptic soy broth (TSB) or in tryptic soy agar (BD Difco) supplemented with 3% horse blood and where appropriate with antibiotics (Santoro et al. 2010;Iannelli et al. 2021).Escherichia coli was grown in Luria-Bertani broth (LB) or LB supplemented with 1.5% agar (LBA).When required, 20 µg/ml chloramphenicol was added to both LB and LBA media.Bacteria for Western blot, Dot blot, Flow cytometry and Immunofluorescence microscopy analysis were grown in TSB without dextrose (BD Difco) until the late exponential phase (OD 590 = 0.3, corresponding to approximately 10 6 CFU/ml).Nisin for induction was extracted as already described (Xiao et al. 2010).Briefly, 100 mg of 2.5% nisin (Sigma-Aldrich) was resuspended in 50 ml of 50% ethanol in distilled water, stirred for 2 h before centrifugation at 1,500 x g for 5 min, and supernatant was then collected and stored at -20°C.Nisin sensitivity was determined by MIC assays as already described (Fox et al. 2021).Briefly, bacteria were grown in TSB until reaching the exponential phase (OD 590 = 0.3, corresponding to approximately 10 8 CFU/ ml), then culture aliquots were diluted 1:100 in TSB (10 6 CFU/ml) and 100 µl were added to a 96-wells microplate containing 100 µl of serial twofold dilutions of nisin, reaching a final concentration of 5 × 10 5 CFU/ml in each well.Plates were incubated at 37°C and visually analyzed after 1 3 18 h.Bacterial growth was assessed using the microplate ELISA reader VERSAmax (Molecular Devices).The nisin MIC value for the S. pneumoniae recombinant FR372 strain was 1 µg/ml.For nisin treatment, bacterial cultures grown in TSB until mid-exponential phase (OD 590 = 0.3) were split in 5 different aliquots of which 4 were treated with 3-fold serially diluted nisin concentrations (from 27 to 1 ng/ml).Cultures were incubated at 37°C for 60 min, then bacterial cells were harvested by centrifugation at 10,000 x g at 4°C for 5 min and stored at -70°C in phosphate-buffered saline (PBS) containing 10% glycerol.

Vectors construction
Standard molecular cloning procedures were used.Briefly, the restriction reactions mixture contained, in a final volume of 50 µl, 1× buffer, 1 U enzyme, and 1 µg plasmid DNA.Mixture was incubated at 37°C for 60 min, then enzyme was inactivated at 65°C for 20 min.If necessary, mixture was purified with the Quick-DNA purification kit according to manufacturer's instructions (Zymo Research).Digested DNA was visualized and quantified by both electrophoretic and spectrophotometric analyses.The sequence of vectors This study a comC encodes the competence stimulating peptide precursor (Pozzi et al. 1996;Iannelli et al. 2005); emm6.1 encodes the S. pyogenes M6 surface protein (Fischetti et al. 1988); aphIII confers resistance to kanamycin; erm(B) and erm(C) confer resistance to erythromycin (Cassone et al. 2006); str-41 and str-204 indicate point mutations conferring resistance to streptomycin; Cm, chloramphenicol; Sm, streptomycin; Tc, tetracycline; Km, kanamycin; Em, erythromycin; Amp, ampicillin; Fus, fusidic acid; Rif, rifampicin to 8,007 of Tn5251 sequence (GenBank no.FJ711160.1).
Vector pSMB47 was purified from E. coli GP394, digested with BamHI and HindIII restriction enzymes and ligated to a 2,322-bp synthetic DNA fragment containing the L. lactis nisRK operon (2,227 bp, nts 305,189 to 307,415 GenBank no.CP002094) joined to the S. pneumoniae dexB terminator (71 bp, nts 380 to 450 GenBank no.AF026471.3).The fragment, customized by DNA2.0 ATUM company, is flanked by HindIII on one end and by BamHI, MluI, BglII on the was verified by PCR followed by Sanger sequencing as reported (Iannelli et al. 1998).Insertion vector pSMB47 (GenBank no.U69267) contains the cat and erm(B) genes conferring resistance to chloramphenicol and erythromycin allowing for selection in E. coli and gram-positive bacteria, respectively (Fig. 1) (Manganelli et al. 1998).The vector contains two non-contiguous fragments of the ICE Tn5251.
The fragments are 1,351 bp and 1,028 bp in length and correspond to nucleotides (nts) 4,080 to 5,430 and 6,980 at 42°C for 2 min and placed back on ice for 5 min; volume was adjusted to 1 ml with LB containing 10% glycerol, the mixture was incubated at 37°C with shaking at 180 RPM for 180 min and then frozen at -70°C.Recombinant strains were selected by spreading 0.01 ml of the transformation mixture onto LBA plates containing the appropriate antibiotic.

Mating assays
The protocol for plate mating experiments was already described (Iannelli et al. 2021).Briefly, donor and recipient cells were grown separately in TSB in the presence of the appropriate antibiotic at 37°C, until the end of the exponential phase (OD 590 = 0.8).Cells were mixed at a 1:10 ratio, harvested by centrifugation at 3,000 x g for 15 min, resuspended in 0.1 ml of TSB and plated on TSA plates enriched with 5% horse blood.After 4 h of incubation in 5% CO2 at 37°C, cells were harvested by scraping the plate with a sterile swab and resuspended in 1 ml TSB containing 10% glycerol.Transconjugants were selected with a multilayer plating procedure.

DNA and RNA purification
Plasmid DNA purification was carried out using the NucleoSpin Plasmid Kit (Macherey-Nagel) following manufacturer's instructions.Briefly, E. coli cells were cultured in LB medium until reaching OD 590 = 3.0, harvested by centrifugation, lysed and loaded on the column.DNA was eluted in 30 µl of 5 mM Tris/HCl pH 8.5.Purified DNA was quantified by both electrophoretic and spectrophotometric analyses.Total RNA was purified from bacterial cultures using the NucleoSpin RNA Isolation Kit (Macherey-Nagel) according to manufacturer's instructions.Purified RNA was subjected to amplification grade DNase I treatment (AMPD1, Sigma-Aldrich).The 10 µL reaction mixture was in 1× reaction buffer, containing 1 unit of DNase I and 500 ng of RNA preparation, and was incubated at 37°C for 15 min.EDTA stop solution was added at a final concentration of 5 mM, while DNase I was inactivated by heating at 70°C for 10 min.Agilent 2100 Bioanalyzer (Agilent Technologies) apparatus and the Agilent RNA 6000 Nano Kit (Agilent Technologies) were used to evaluate RNA integrity.

Reverse transcription and quantitative real-time PCR
Reverse transcription for cDNA synthesis was carried out using the

Preparation of competent cells and transformation
S. pneumoniae competent cells preparation and transformation were carried out as reported (Iannelli and Pozzi 2004).
Frozen pneumococcal competent cells were thawed and incubated at 37°C for 10 min in the presence of 100 ng/ ml competence stimulating peptide (competence induction).
Then transforming DNA was added at a final concentration of 1 µg/ml and the mixture was incubated at 30°C for 20 min (transformation reaction).Prior to transformation, DNA vectors pMBT5 and pMBT6 were linearized with KpnI and SacI, respectively.Selection of recombinant strains was obtained by a multilayer plating procedure (Iannelli et al. 2021).The frequency of transformation was 3.4 × 10 − 2 transformants/total CFU and 5.4 × 10 − 2 transformants/total CFU when pMBT5 or pMBT6 was used as donor DNA, respectively.E. coli competent cells were obtained as follows: an overnight culture was diluted 100-fold in 200 ml of LB medium and incubated at 37°C with shaking at 180 RPM; upon reaching an OD 590 = 0.6, cells were centrifuged at 5,000 x g for 10 min at 4°C in 50-ml tubes; each pellet was gently resuspended in 25 ml ice-cold 0.1 M MgCl 2 solution and centrifuged at 5,000 x g at 4°C for 10 min; finally, the cell pellet was resuspended in 5 ml of an ice-cold 0.1 M MgCl 2 solution supplemented with 10% glycerol, aliquoted and frozen at -70°C (Chang et al. 2017).Transformation of E. coli competent cells was performed as follows: 0.2 ml of competent cells containing the transforming DNA at a final concentration of 0.1 µg/ml were incubated in an ice bath for 10 min swirling gently every 2 min, heat shocked GenBank accession no.FJ711160.1),respectively, were used to amplify the integrated genetic construct, whereas already described primer pairs directed to Tn5253 and chromosomal flanking regions (Iannelli et al. 2014;Santoro et al. 2018Santoro et al. , 2021) ) were used to confirm structure and chromosomal integration of the recombinat Tn5253::[nisRK]-[emm6::ha1] element.

Western blot analysis
A total of 10 7 colony forming units (CFUs) was incubated at 37°C for 60 min in 200 µl of Protoplasting Buffer (20% Raffinose, 50 mM Tris-HCl pH 8.0, 50 mM EDTA) containing 4 mg/ml lysozyme (Colombini et al. 2023b).The protoplasts were sedimented by centrifugation at 5,000 x g for 5 min and the supernatant was collected.To concentrate the supernatant, 4 volumes of acetone were added and the mixture was incubated at -20°C for 60 min, then centrifuged at 15,000 x g for 10 min.The pellet was air-dried for 15 min and resuspended in 50 µl of 10 mM Tris, 1 mM EDTA, 2% SDS buffer.Cell wall extract was quantified using the Qubit Protein Assay Kit (ThermoFisher), according to manufacturer's instructions, washed in ice-cold PBS and resuspended in 1× NuPAGE LDS Sample Buffer and 1× NuPAGE Sample Reducing Agent, incubated at 70°C for 10 min, then separated on 4-12% NuPAGE Bis-Tris Protein Gel on a XCell SureLock Mini-Cell device following manufacturer's instructions (ThermoFisher).The gel was dry transferred onto nitrocellulose membrane (Ther-moFisher) using the iBlot Dry Blotting System apparatus (ThermoFisher) and blocked with 1× Tris Buffered-Saline (TBS) (150mM NaCl, 20 mM Tris-Cl, pH 7.5) containing 0.05% Tween20 (Sigma-Aldrich) and 5% nonfat dry milk (Applichem) for 2 h at room temperature (RT) on a rocking platform.The membrane was incubated at 4°C overnight in the presence of anti-M6 rabbit serum, diluted 1:1,000 in 1× TBS, 0.05% Tween20, 3% milk.The membrane was washed 3 times for 10 min in 1× TBS, 0.05% Tween20, and then incubated for 60 min at RT in the presence of goat anti-rabbit IgG Alkaline-Phosphatase conjugated antibody (Sigma-Aldrich) diluted 1:10,000 in 1× TBS, 0.05% Tween20, 3% milk.The membrane was washed twice in 1× TBS 0.05% Tween20 3% milk for 3 min and once in 1× TBS for 15 min.Proteins were detected using the colorimetric detection of Alkaline-Phosphatase labeled molecules method.Briefly, 1,65 mg/ml 5-bromo-4-chloro-3-indolyphosphate-p-toluidine (BCIP) salt and 3.3 mg/ml nitro-blue tetrazolium chloride (NBT) chromogen (Sigma-Aldrich) were added to 100 mM Tris, 100 mM NaCl and 5 mM MgCl 2 pH 9.5 working solution, according to standard procedures.The membrane was incubated in this substrate buffer until color total RNA was incubated at 65°C for 10 min (denaturation step) and placed on ice.Then, a 7 µl mixture containing 1× Transcriptor Reverse Transcriptase Reaction buffer, 20 U Protector RNase Inhibitor, 1 mM Deoxynucleotide Mix and 10 U Transcriptor Reverse Transcriptase, was added.The 20 µl mixture was incubated at 25°C for 10 min, followed by 60 min at 50°C, inactivated at 85°C for 5 min and finally cooled on ice.Real-time PCR mixture contained, in a final volume of 20 µl, 1× KAPA SYBR FAST qPCR reaction mix, 5 pmol of each primer and 1 µl of cDNA.Thermal profile was an initial 3 min denaturation step at 95°C followed by 40 cycles of repeated denaturation (0 s at 95°C), annealing (20 s at 60°C), and polymerization (45 s at 72°C).
The temperature transition rate was 20°C/s in the denaturation and annealing step and 5°C/s in the polymerization step.Oligonucleotide primers targeting the emm6.1::ha1gene were IF994 (5 Serial dilutions of the S. pneumoniae FR24 chromosomal DNA with known concentration were used to build a standard curve for the gyrB gene by plotting the threshold cycle against the number of chromosome copies (Lazzeri et al. 2012;Santoro et al. 2023).This standard curve was recalled in the instrument software to standardize the emm6.1::ha1transcript copy number.To discriminate desired amplification products from primer-dimer products, the melting curves were analysed.As an internal expression control we used the housekeeping gyrB gene.

Nanopore sequencing
High-molecular-weight genomic DNA was purified using a raffinose-based method as reported (Teodori et al. 2021;Pinzauti et al. 2022b).Briefly, a bacterial cell-wall enzymatic digestion was performed followed by protoplast osmotic lysis and DNA purification with Sevag (chloroform-isoamyl alcohol, 24:1 [vol: vol]).Purified genomic DNA was size-selected with 0.5× AMPure XP beads (Beckman Coulter) and used for Nanopore sequencing libraries preparation with the SQK-LSK108 kit (Oxford Nanopore Technologies) following the manufacturer's instructions.Sequencing runs were managed by a GridION X5 device (Oxford Nanopore Technologies) as previously described (Pinzauti et al. 2022a).

Long PCR and sanger sequencing
Long PCRs and direct PCR sequencing were carried out as described (Iannelli et al. 1998).

The primer pair IF394 (5'-G C T A T A G T A T A A G C C A T A C T T-3') and IF668 (5'-G T T G T G A T T G C C T T G T G G G T-3') directed at the tet(M)
and orf13 (nts 4,387 -4,407 and 7,092 -7,111 of Tn5251, goat anti-rabbit IgG secondary antibody (Sigma) at 37°C for 30 min in a humidified chamber.Slide were washed three times with PBS, DAPI II counterstain (Abbot) was added at a final concentration of 12.5 pg/ml, washed again three times with PBS, air-dried, and covered with cover slip.Bacteria were observed under a Leica 6500 fluorescence microscope (Leica) using a 100x oil objective.Images were captured with a CFTR6500 digital camera (Leica).

Construction of a nisin-inducible chromosomal gene expression system based on ICE Tn5253
We constructed the insertion vector pMBT5, a derivative of pSMB47 (Manganelli et al. 1998), that integrates by double crossover recombination in Tn5251, an ICE contained in the composite element Tn5253 and carrying the tetracycline resistance tet(M) gene (Fig. 1).The vector contains two Tn5251 homologous regions to drive recombination, the L. lactis two-component regulatory system nisRK joined to the S. pneumoniae dexB terminator, and a multiple cloning site suitable for target gene insertion under control of the L. lactis nisin inducible promoter PnisA (Kuipers et al. 1993).Subsequently, a DNA fragment containing the fusion gene emm6.1::ha1joined to the PnisA promoter region was cloned in vector pMBT5, yielding vector pMBT6 (Fig. 1).Competent cells of S. pneumoniae FR24 containing Tn5253 integrated into the chromosome, were transformed with a linearized pMBT6 vector (Fig. 2).Homologous recombination promotes integration of the genetic construct [nisRK]-[emm6.1::ha1] along with the erm(B) and cat resistance markers and the deletion of 1,549 bp including the first 480 nucleotides of the tet(M) coding sequence.Selection of recombinant strains was obtained by acquisition of erythromycin resistance and loss of tetracycline resistance.Genome sequence analysis of the representative erythromycin-resistant transformant FR372 confirmed the presence of the recombinant ICE Tn5253:: Genetic stability of the resistance phenotype was tested by growing pneumococcal cells in liquid medium without antibiotics for 220 generations.After plating on a non-selective medium, each of the 300 randomly picked colonies was resistant to erythromycin.PCR and sequencing carried out on 10 out 300 isolates confirmed that the genetic construct was intact and correctly integrated.The effect of the chromosomal integration of the construct on growth rate and on transformation frequency was investigated in the FR372 transformant and its FR24 parental strain.Both strains showed comparable duplication time (1 generation/hour) and transformation frequency (2% of total CFU).
development appeared and the reaction was stopped washing the membrane with distilled water.

Dot blot analysis
Bacterial cells were spotted onto a nitrocellulose membrane with a Bio-Dot microfiltration blotting apparatus (Bio-Rad) using a vacuum-manifold procedure.Sample spotting was performed in two-fold serial dilutions from 1 × 10 7 to 7.8 × 10 4 CFU.Uninduced recombinant strains and their parental strains were spotted as controls.The membrane was dried at 80°C and blocked with TBS containing 0.05% Tween20 and 5% nonfat dry milk for 2 h at RT, followed by an overnight incubation at 4°C with rabbit anti-HA1 antibody (Sino Biological) diluted 1:1,000 in 1× TBS, 0.05% Tween20, 3% milk.The membrane was washed 3 times for 3 min with 1× TBS, 0.05% Tween20 and incubated for 60 min at RT with goat anti-rabbit IgG Alkaline-Phosphatase conjugated antibody (Sigma-Aldrich) diluted 1:10,000 in 1× TBS, 0.05% Tween20, 3% milk.The membrane was washed again 3 times for 3 min with 1× TBS, 0.05% Tween20 and once with TBS, for 15 min.For standard curve building, known quantities of the purified HA1 (Sino Biological) protein ranging from 25 ng to 195 pg were used.
Protein quantification was obtained by densitometric analysis using the ImageJ v.1.53esoftware.

Flow cytometry
A total of 10 7 CFUs were washed in ice cold PBS and blocked in PBS-BSA 2% at 37°C for 30 min.Next, cells were incubated with primary anti-HA1 antibody (Sino Biological, 11055-RM10) diluted 1:160 in PBS-BSA 2% at 4°C for an hour, washed twice in PBS and incubated with 1:160 diluted secondary, FITC-conjugated, anti-rabbit IgG antibody (Sigma-Aldrich) at 37°C for 20 min.Cells were washed in PBS and fixed with BD Cytofix/Cytoperm solution (Becton Dickinson) at 4°C for 15 min.Samples were finally resuspended in 300 µl of filtered PBS and analysed using FACS scan instrument (Becton Dickinson).

Immunofluorescence microscopy
S. pneumoniae FR372 cells, induced with nisin 27 mg/ml or not induced, were grown until late expenential phase, washed and resuspended in PBS.A total of 10 µl, corresponding to approximately 10 6 CFU, were spotted on a glass slide and air-dried.Bacteria were fixed for 5 min in methanol and washed in PBS.The slide was incubated at 4°C for 1 h in a humidified chamber with anti-M6 rabbit serum, diluted 1:25 in PBS-BSA 2%.Then, cells were washed three times with PBS and incubated with a fluorochrome FITC-conjugated An estimated 69 kDa reactive band, corresponding to the predicted M6-HA1 protein size, was detected when using anti-M6 rabbit serum (Fig. 4A).Semi-quantitative analysis of M6-HA1 expression was carried out by Dot blot on cell extracts using an anti-HA1 rabbit monoclonal antibody.M6-HA1 protein quantities ranged from 291 to 772 ng per 10 9 CFU of induced pneumococcal cultures (Fig. 4B).Flow cytometry analysis performed on live bacterial cells showed the presence of the M6-HA1 protein on the pneumococcal surface (Fig. 4C).The increase in fluorescence intensity observed in FR372 compared with the negative control was correlated with nisin concentration.The presence of the M6-HA1 protein on pneumococcal surface was confirmed by Immunofluorescence microscopy analysis (Fig. 5).

Evaluation of the nisin-inducible gene expression system in Streptococcus pneumoniae
Pneumococcal cultures of the FR372 recombinant strain in the exponential growth phase were treated with subinhibitory doses of nisin (from 1 to 27 ng/ml), and total RNA purified from treated and untreated control cultures was used as template for detection and quantification of the emm6.1::ha1transcript.A specific emm6.1::ha1transcript was found in both treated and untreated cultures, and the transcript copy number increased with the growing nisin concentration used for induction (Fig. 3A).The levels of gene expression ranged from 5.12 × 10 3 ± 6.29 × 10 2 to 2.83 × 10 6 ± 1.90 × 10 6 copies per nanograms of total RNA, obtained from either untreated or nisin treated (27 ng/ml) cultures.

Detection and quantification of the induced protein production in S. pneumoniae
The expression of M6-HA1 fusion protein was analyzed by Western blot, Dot blot, Flow cytometry and Immunofluorescence microscopy.Western blot analysis was performed on cell extracts of S. pneumoniae FR372 cultures induced with a nisin concentration ranging from 1 to 27 ng/ml.

Discussion
In this study we developed and validated a nisin-inducible chromosomal host-vector expression system based on ICE Tn5253 of S. pneumoniae and transferable in a variety of streptococci and enterococci.The system is constituted of a shuttle vector capable of replication in E. coli but not in S. pneumoniae, where it integrates in Tn5251, a Tn916family ICE contained in the composite element Tn5253.The insertion vector carries the L. lactis two-component regulatory system nisRK and a multiple cloning site for conjugation frequencies ranging from 8.1 × 10 − 7 to 2 × 10 − 5 transconjugants per donor in E. faecalis and S. gordonii, respectively.Representative transconjugants were tested for genetic stability of the recombinant element, and used for further analysis.The emm6.1::ha1 transcript was detected and quantified by RT-qPCR (Fig. 3B) and Western blot analysis confirmed the presence of the M6-HA1 protein in all bacterial species (Fig. 6).Furthermore, M6-HA1 protein was quantified using Dot blot analysis, whereas protein surface expression was detected by Flow cytometry.M6-HA1 protein quantities ranged from a minimum of about 290 ng in S. gordonii to a maximum of about 600 ng in E. faecalis upon induction with 27 ng/ml of nisin (Figure S2).Accordingly, Flow cytometry analysis confirmed the presence of M6-HA1 protein on the cell surfaces in all induced al. 2011).On the other hand, when using integrative vectors for bacterial manipulation, the choice of a transcriptionally neutral chromosomal site represents a major drawback (Guiral et al. 2006b;Keller et al. 2019).Our genetic system allows to overcome this limitation by using the natural interaction between Tn5253 and its bacterial host.Tn5253 integration site is located in the conserved essential rbgA gene (Santoro et al. 2021), thus the expression system has the potential to transfer to a wide range of bacterial species.
In fact, the system was efficiently transferred by conjugation in different streptococcal species including S. gordonii, S. pyogenes, S. agalactiae and E. faecalis, where a nisin doseresponse relationship was also detected.Tn916, a broad host range conjugative transposon, was previously used for the construction of ICE-based host-vector expression systems for pathogenic, non transformable bacteria (Mullany et al. 1994(Mullany et al. , 2012;;Manganelli et al. 1998;McBride and Sonenshein 2011a, b).Tn5251 is highly homologous to Tn916 and generally transfers 'hitchhiking' Tn5253 conjugation machinery, but it is also capable of independent conjugal transfer in different bacterial species (Santoro et al. 2010).
This characteristic further expands the potential bacterial host range where the Tn5253-based nisin-inducible expression system can be used.In conclusion, we constructed a transferable ICE Tn5253-based host-vector system tested for controlled heterologous gene expression and potentially usable for a wide range of applications including complementation assays and/or physiological studies in S. pneumoniae and other transformable and non transformable bacteria including pathogenic species otherwise difficult to manipulate.
target gene insertion under the control of the L. lactis nisin inducible promoter PnisA.For system validation, we cloned the fusion gene emm6.1::ha1encoding the fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin.The emm6.1::ha1 was previously cloned in our S. gordonii host-vector system under a strong constitutive promoter with efficient M6-HA1 fusion protein expression on bacterial surface (Iannelli et al., unpublished).
Here, we observed a tunable regulation of M6-HA1 protein expression when inducing bacterial cultures with various concentrations of nisin, making this expression system suitable for mucosal vaccine studies requiring fine adjustement of antigen dose.A basal gene expression was found in the untreated control culture as already reported (Eichenbaum et al. 1998).Widely used plasmid-based expression systems are capable of autonomous replication in the host, resulting in multiple plasmid copies that can lead to perturbations of the cell physiology (Friehs 2004).Since Tn5253 integrates at a single, specific site into the bacterial chromosome (Santoro et al. 2021) and replicates passively during chromosomal duplication (Johnson and Grossman 2015), our ICE-based expression system ensures a single-copy integration of the target gene.Furthermore, plasmids can be lost if selection is not maintained over generations, while ICEs are vertically transmitted to the bacterial daughter cells.In fact, genomic analysis of the population structure of S. pneumoniae highlighted that ICEs are conserved within lineage suggesting that a stable integration into the chromosome ensures genetic maintenance of these mobile elements (Croucher et

Fig. 1
Fig. 1 Schematic representation of pMBT6 vector construction.Insertion vector pSMB47 contains: (i) the chloramphenicol resistance cat and erythromycin resistance erm (B) genes allowing for selection in E. coli and gram-positive bacteria, respectively; and (ii) two noncontiguous fragments of the ICE Tn5251 (striped red boxes) driving integration in the element.The 2,322-bp synthetic construct containing the L. lactis nisRK two-component regulatory system was inserted

Fig. 2
Fig. 2 Schematic representation of the S. pneumoniae ICE Tn5253based nisin-inducible gene expression system construction.The insertion vector pMBT6 contains: (i) the L. lactis nisRK two-component regulatory system; (ii) the emm6.1::ha1fusion gene under the control of the L. lactis PnisA promoter; and (iii) two flanking DNA fragments (striped red boxes) homologous to the ICE Tn5251 which is integrated Figure is not drawn to scale transconjugants with a positive shift slightly increasing in a nisin dose-dependent manner.

Fig. 3 Fig. 5
Fig.3Emm6.1::ha1fusion gene expression upon nisin treatment in S. pneumoniae (A) and in other streptococci and enterococci (B).RT-qPCR was performed on total RNA purified from the recombinant strains using primers targeting the emm6.1::ha1fusion gene.Gene expression level was calculated as copy number per nanograms of

Fig. 6
Fig. 6 M6-HA1 fusion protein expression analysis in other streptococci and enterococci.M6-HA1 identification was obtained by Western blot carried out on recombinant bacterial cell extracts from cultures induced with 3-fold serially diluted nisin concentrations.Samples were

Table 1
R , Rif R , Em R , Cm R